Project Summary Iron-sulfur (Fe-S) cluster assembly in mitochondria is an essential process for all eukaryotic cells because Fe-S cluster proteins/enzymes are involved in myriad processes including oxidative phosphorylation, DNA repair, protein translation, metabolic conversions, and regulation of iron and oxygen. The formation of Fe-S clusters in mitochondria involves building of the Fe-S cluster intermediate on a scaffold protein, Isu1, and then transferring it to recipients. The sulfur for Fe-S clusters derives exclusively from the cysteine desulfurase Nfs1/Isd11, which binds the substrate cysteine, forms a persulfide, and transfers the persulfide sulfur to Isu1. The process is highly regulated. The consequences of misregulation are pathology and diseases, but knowledge of the regulatory controls is very limited. We have found that Nfs1 can be phosphorylated in mitochondria and that this enhances its activity. The focus of Aim 1 is on mechanistic studies of how phosphorylation alters the Nfs1 cysteine desulfurase activity, and how this relates to similar effects by frataxin. Frataxin is a disease gene implicated in Friedreich's ataxia, and it is also a positive regulator of cysteine desulfurase activity. The roles of the pleiotropic Yck2 kinase (and its paralog Yck1) will be defined in terms of the ?eclipsed? protein localization in the mitochondrial matrix and effects on Fe-S cluster assembly. We have strong evidence for the role of a GTPase and GTP hydrolysis in transfer of the Nfs1-bound persulfide sulfur to the scaffold protein. Aim 2 is to identify the GTPase involved in Fe-S cluster synthesis in mitochondria. The GTPase activity associated with an Fe-S cluster assembly complex has been purified from mitochondria and a nucleotide-GTPase adduct has been detected, leading us to believe we have the GTPase in hand. The site in Fe-S cluster assembly where the GTPase operates will be ascertained, and expected genetic relationships with the mitochondrial GTP/GDP carrier, Ggc1, will be probed. Aim 3 builds on the recent breakthrough in the field identifying the mitochondrial acyl carrier protein, Acp1, as a key player in Fe-S cluster assembly (Van Vranken et al. eLife 2016; 5:e17828). The role of Acp1 in bringing Nfs1 and Isd11 together to activate the enzyme and to stabilize Nfs1 against denaturation and/or degradation will be examined in Aim 3. Iron transporters Mrs3 and Rim2 also activate and stabilize Nfs1 in Acp1-depleted mitochondria, and the iron connection will be explored. Finally, Aim 4 pertains to a form of the Nfs1 cysteine desulfurase held in the mitochondrial inner membrane in inactive form that can be activated by Acp1 in concert with other matrix protein(s). The goal is to identify the other matrix protein(s) that activate Nfs1, and also the membrane proteins that repress it. The proposed work will be performed in yeast because of the availability of genetic tools, deletion strains, plasmid libraries, and detailed databases. In the field of Fe-S cluster assembly studies, yeast discoveries have generally anticipated discoveries in mammalian cells, and yet components are conserved and orthologous.